The APC Subunit Doc1 Promotes Recognition of the Substrate Destruction Box  Christopher W. Carroll, Maria Enquist-Newman, David O. Morgan  Current Biology  Volume 15, Issue 1, Pages 11-18 (January 2005) DOI: 10.1016/j.cub.2004.12.066

Figure 1 Doc1 Mutants Are Defective in Processivity (A) The position of highly conserved residues identified by structure-based sequence alignments is shown on budding-yeast Doc1 [15, 16]. Residues that we show are required for Doc1 function are labeled in red. This image was generated with CHIMERA [20]. (B) Doc1 mutants have defects in processivity. APC was immunoprecipitated with anti-Cdc26 antibodies from ∼1 mg of extract prepared from strain TC92 (MAT a, bar1, doc1Δ::URA3) expressing wild-type DOC1 (Wt) or various mutants from the DOC1 promoter on a CEN/ARS plasmid (see Experimental Procedures). Control cells (vector) express no Doc1. Immunoprecipitates were washed three times in lysis buffer and divided in two. One half (top panel) was used for measurement of cyclin ubiquitination activity and visualized with a PhosphorImager. The other half (bottom panels) was used for Western blotting of the APC subunits Cdc16 and Doc1. (C) Quantification of ubiquitination reactions like those in (B). The numbers of mono (cyclin-ub1)-, di (cyclin-ub2)-, and tri (cyclin-ub3)-ubiquitinated species in each lane were quantified, and the processivity of the reaction was determined by calculating the ratio of ubiquitin to cyclin in the reaction products. Because samples containing fully active Doc1 generate some products above the tri-ubiquitinated species, the data presented are likely an underestimate of the actual ratio in some cases. Error bars represent the standard deviation from three independent experiments. Current Biology 2005 15, 11-18DOI: (10.1016/j.cub.2004.12.066)

Figure 2 Functional Analysis of Doc1 Mutations that Affect Processivity (A and B) Doc1 mutants fall into two classes. Increasing concentrations (up to 12 μM) of purified wild-type or Doc1 mutant proteins were added to ∼1 nM APCdoc1Δ in complete ubiquitination reactions. MBP alone was used as a negative control. Reactions were allowed to proceed until ∼5% of the total substrate was converted to product, and the processivity of the reaction was quantified as described in Figure 1C. Data were fitted to a rectangular hyperbola (solid line). Error bars represent the standard deviation from three independent experiments. ([A], bottom right) The small graph is a scaled version of the large graph and includes additional data at high (>1 μM) Doc1 concentrations. (C) doc1-4A can bind but not activate the APC. Increasing concentrations of the doc1-4A mutant protein were added to ∼1 nM APCdoc1Δ in the presence of ∼10 nM wild-type Doc1. Reactions were analyzed as above. Solid line is the best fit of the data. Error bars represent the standard deviation from three independent experiments. The dashed line represents the average processivity of APCdoc1Δ in ubiquitination reactions lacking recombinant Doc1. Current Biology 2005 15, 11-18DOI: (10.1016/j.cub.2004.12.066)

Figure 3 Doc1-4A Mutants Are Defective in Ubiquitination of Multiple Substrates (A) The ligand binding region of Doc1 is not required for APC assembly. APC was purified in parallel from strains TC130 (MAT α, CDC16::CDC16-TAP:HIS3, doc1Δ::URA3, trp1::TRP1:DOC1) and TC131 (MAT α, CDC16::CDC16-TAP:HIS3, doc1Δ::URA3, trp1::TRP1:doc1-4A) with the TAP method [17]. Equal volumes of the final eluate (∼20 μl) from the wild-type (Wt) or mutant (4A) APC purifications were separated on a 12.5% polyacrylamide gel, and the proteins were visualized by silver staining. The identity of individual APC subunits is shown on the right; molecular weight is indicated to the left. The doc1-4A mutant protein migrates slightly faster than wild-type Doc1 under these conditions. (B) APCdoc1-4A is defective for processive substrate ubiquitination in vitro. Complete ubiquitination reactions containing equal amounts (∼1 nM final concentration) of wild-type APC (Wt), APCdoc1Δ (doc1Δ), or APCdoc1-4A (doc1-4A) were performed in parallel with the indicated substrates; control reactions (–) have no APC. All substrates were produced in vitro by coupled transcription and translation and are labeled with 35S-methionine. Reactions containing sea urchin cyclin B were resolved on a 15% acrylamide gel. All others were resolved on 7.5% acrylamide gels. Current Biology 2005 15, 11-18DOI: (10.1016/j.cub.2004.12.066)

Figure 4 The Substrate D-box Is Required for Recognition by Doc1 Hsl1 (A), Pds1 (B), and Clb2 (C), each containing intact targeting motifs or mutations in the D-box, KEN-box, or both, were included in complete ubiquitination reactions containing no APC (–), wild-type APC (Wt), or APCdoc1-4A (4A). Reactions were performed as in Figure 3, and identical results were obtained in three independent experiments. (+) represents wild-type sequence; (–) represents a mutation. D-box mutations are (RxxLxxxxN → AxxAxxxxA, except Pds1, which is RxxL → AxxA); KEN-box mutations are (KEN → AAA for Clb2 and Pds1, KENxxxE → AAAxxxA for Hsl1). Current Biology 2005 15, 11-18DOI: (10.1016/j.cub.2004.12.066)

Figure 5 Doc1 Is Required for APC Activity In Vivo (A and B) The metaphase-to-anaphase transition is delayed in doc1-4A cells. Strains TC136 (MAT a, bar1, doc1Δ::URA3, leu2::LEU2:DOC1, PDS1::PDS1-13myc:HIS3) and TC137 (MAT a, bar1, doc1Δ::URA3, leu2::LEU2:doc1-4A, PDS1::PDS1-13myc:HIS3) were arrested with α factor (1 μg/ml) and released. Samples were taken every half hour and analyzed for cell cycle progression. Budding index (A) and DNA segregation (B) in wild-type (Wt) or doc1-4A mutant cells were quantified as a function of time after release from G1 arrest. Data are from a representative experiment that was repeated three times. Over 100 cells were counted for each time point. (C) APC substrates are stabilized in doc1-4A cells. 10 μg of cell lysate from each time point was resolved on a 10% acrylamide gel and transferred to nitrocellulose for Western blotting of the indicated APC substrate. Current Biology 2005 15, 11-18DOI: (10.1016/j.cub.2004.12.066)